Experimental and modelling investigation of monolayer development with clustering

Standard differential equation–based models of collective cell behaviour, such as the logistic growth model, invoke a mean–field assumption which is equivalent to assuming that individuals within the population interact with each other in proportion to the average population density. Implementing such assumptions implies that the dynamics of the system are unaffected by spatial structure, such as the formation of patches or clusters within the population. Recent theoretical developments have introduced a class of models, known as moment dynamics models, which aim to account for the dynamics of individuals, pairs of individuals, triplets of individuals and so on. Such models enable us to describe the dynamics of populations with clustering, however, little progress has been made with regard to applying moment dynamics models to experimental data. Here, we report new experimental results describing the formation of a monolayer of cells using two different cell types: 3T3 fibroblast cells and MDA MB 231 breast cancer cells. Our analysis indicates that the 3T3 fibroblast cells are relatively motile and we observe that the 3T3 fibroblast monolayer forms without clustering. Alternatively, the MDA MB 231 cells are less motile and we observe that the MDA MB 231 monolayer formation is associated with significant clustering. We calibrate a moment dynamics model and a standard mean–field model to both data sets. Our results indicate that the mean–field and moment dynamics models provide similar descriptions of the 3T3 fibroblast monolayer formation whereas these two models give very different predictions for the MDA MD 231 monolayer formation. These outcomes indicate that standard mean–field models of collective cell behaviour are not always appropriate and that care ought to be exercised when implementing such a model.

Choosing an appropriate modelling framework for analysing multispecies co-culture cell biology experiments

In vitro cell biology assays play a crucial role in informing our understanding of the migratory, proliferative and invasive properties of many cell types in different biological contexts. While mono-culture assays involve the study of a population of cells composed of a single cell type, co-culture assays study a population of cells composed of multiple cell types (or subpopulations of cells). Such co-culture assays can provide more realistic insights into many biological processes including tissue repair, tissue regeneration and malignant spreading. Typically, system parameters, such as motility and proliferation rates, are estimated by calibrating a mathematical or computational model to the observed experimental data. However, parameter estimates can be highly sensitive to the choice of model and modelling framework. This observation motivates us to consider the fundamental question of how we can best choose a model to facilitate accurate parameter estimation for a particular assay. In this work we describe three mathematical models of mono-culture and co-culture assays that include different levels of spatial detail. We study various spatial summary statistics to explore if they can be used to distinguish between the suitability of each model over a range of parameter space. Our results for mono-culture experiments are promising, in that we suggest two spatial statistics that can be used to direct model choice. However, co-culture experiments are far more challenging: we show that these same spatial statistics which provide useful insight into mono-culture systems are insuffcient for co-culture systems. Therefore, we conclude that great care ought to be exercised when estimating the parameters of co-culture assays.

Ion-assisted functional monolayer coating of nanorod arrays in hydrogen plasmas

Uniformity of postprocessing of large-area, dense nanostructure arrays is currently one of the greatest challenges in nanoscience and nanofabrication. One of the major issues is to achieve a high level of control in specie fluxes to specific surface areas of the nanostructures. As suggested by the numerical experiments in this work, this goal can be achieved by manipulating microscopic ion fluxes by varying the plasma sheath and nanorod array parameters. The dynamics of ion-assisted deposition of functional monolayer coatings onto two-dimensional carbon nanorod arrays in a hydrogen plasma is simulated by using a multiscale hybrid numerical simulation. The numerical results show evidence of a strong correlation between the aspect ratios and nanopattern positioning of the nanorods, plasma sheath width, and densities and distributions of microscopic ion fluxes. When the spacing between the nanorods and/or their aspect ratios are larger, and/or the plasma sheath is wider, the density of microscopic ion current flowing to each of the individual nanorods increases, thus reducing the time required to apply a functional monolayer coating down to 11 s for a 7-μm-wide sheath, and to 5 s for a 50-μm-wide sheath. The computed monolayer coating development time is consistent with previous experimental reports on plasma-assisted functionalization of related carbon nanostructures [B. N. Khare et al., Appl. Phys. Lett. 81, 5237 (2002)]. The results are generic in that they can be applied to a broader range of plasma-based processes and nanostructures, and contribute to the development of deterministic strategies of postprocessing and functionalization of various nanoarrays for nanoelectronic, biomedical, and other emerging applications.

3D cultures of prostate cancer cells cultured in a novel high-throughput culture platform are more resistant to chemotherapeutics compared to cells cultured in monolayer

Despite monolayer cultures being widely used for cancer drug development and testing, 2D cultures tend to be hypersensitive to chemotherapy and are relatively poor predictors of whether a drug will provide clinical benefit. Whilst generally more complicated, three dimensional (3D) culture systems often better recapitulate true cancer architecture and provide a more accurate drug response. As a step towards making 3D cancer cultures more accessible, we have developed a microwell platform and surface modification protocol to enable high throughput manufacture of 3D cancer aggregates. Herein we use this novel system to characterize prostate cancer cell microaggregates, including growth kinetics and drug sensitivity. Our results indicate that prostate cancer cells are viable in this system, however some non-cancerous prostate cell lines are not. This system allows us to consistently control for the presence or absence of an apoptotic core in the 3D cancer microaggregates. Similar to tumor tissues, the 3D microaggregates display poor polarity. Critically the response of 3D microaggregates to the chemotherapeutic drug, docetaxel, is more consistent with in vivo results than the equivalent 2D controls. Cumulatively, our results demonstrate that these prostate cancer microaggregates better recapitulate the morphology of prostate tumors compared to 2D and can be used for high-throughput drug testing.

Ultrasonic wave characteristics of a monolayer graphene on silicon substrate

The present work deals with an ultrasonic type of wave propagation characteristics of monolayer graphene on silicon (Si) substrate. An atomistic model of a hybrid lattice involving a hexagonal lattice of graphene and surface atoms of diamond lattice of Si is developed to identify the carbon-silicon bond stiffness. Properties of this hybrid lattice model is then mapped into a nonlocal continuum framework. Equivalent force constant due to Si substrate is obtained by minimizing the total potential energy of the system. For this equilibrium configuration, the nonlocal governing equations are derived to analyze the ultrasonic wave dispersion based on spectral analysis. From the present analysis we show that the silicon substrate affects only the flexural wave mode. The frequency band gap of flexural mode is also significantly affected by this substrate. The results also show that, the silicon substrate adds cushioning effect to the graphene and it makes the graphene more stable. The analysis also show that the frequency bang gap relations of in-plane (longitudinal and lateral) and out-of-plane (flexural) wave modes depends not only on the y-direction wavenumber but also on nonlocal scaling parameter. In the nonlocal analysis, at higher values of the y-directional wavenumber, a decrease in the frequency band gap is observed for all the three fundamental wave modes in the graphene-silicon system. The atoms movement in the graphene due to the wave propagation are also captured for all the tree fundamental wave modes. The results presented in this work are qualitatively different from those obtained based on the local analysis and thus, are important for the development of graphene based nanodevices such as strain sensor, mass and pressure sensors, atomic dust detectors and enhancer of surface image resolution that make use of the ultrasonic wave dispersion properties of graphene. (C) 2011 Elsevier Ltd. All rights reserved.

Nonlocal continuum mechanics based ultrasonic flexural wave dispersion characteristics of a monolayer graphene embedded in polymer matrix

Wave propagation in graphene sheet embedded in elastic medium (polymer matrix) has been a topic of great interest in nanomechanics of graphene sheets, where the equivalent continuum models are widely used. In this manuscript, we examined this issue by incorporating the nonlocal theory into the classical plate model. The influence of the nonlocal scale effects has been investigated in detail. The results are qualitatively different from those obtained based on the local/classical plate theory and thus, are important for the development of monolayer graphene-based nanodevices. In the present work, the graphene sheet is modeled as an isotropic plate of one-atom thick. The chemical bonds are assumed to be formed between the graphene sheet and the elastic medium. The polymer matrix is described by a Pasternak foundation model, which accounts for both normal pressure and the transverse shear deformation of the surrounding elastic medium. When the shear effects are neglected, the model reduces to Winkler foundation model. The normal pressure or Winkler elastic foundation parameter is approximated as a series of closely spaced, mutually independent, vertical linear elastic springs where the foundation modulus is assumed equivalent to stiffness of the springs. For this model, the nonlocal governing differential equations of motion are derived from the minimization of the total potential energy of the entire system. An ultrasonic type of flexural wave propagation model is also derived and the results of the wave dispersion analysis are shown for both local and nonlocal elasticity calculations. From this analysis we show that the elastic matrix highly affects the flexural wave mode and it rapidly increases the frequency band gap of flexural mode. The flexural wavenumbers obtained from nonlocal elasticity calculations are higher than the local elasticity calculations. The corresponding wave group speeds are smaller in nonlocal calculation as compared to local elasticity calculation. The effect of y-directional wavenumber (eta(q)) on the spectrum and dispersion relations of the graphene embedded in polymer matrix is also observed. We also show that the cut-off frequencies of flexural wave mode depends not only on the y-direction wavenumber but also on nonlocal scaling parameter (e(0)a). The effect of eta(q) and e(0)a on the cut-off frequency variation is also captured for the cases of with and without elastic matrix effect. For a given nanostructure, nonlocal small scale coefficient can be obtained by matching the results from molecular dynamics (MD) simulations and the nonlocal elasticity calculations. At that value of the nonlocal scale coefficient, the waves will propagate in the nanostructure at that cut-off frequency. In the present paper, different values of e(0)a are used. One can get the exact e(0)a for a given graphene sheet by matching the MD simulation results of graphene with the results presented in this article. (c) 2012 Elsevier Ltd. All rights reserved.

Development of a spin polarized low energy electron diffraction system

We have designed and constructed a spin polarized low energy electron diffraction system working in the reflected electron pulse counting mode. This system is capable of measuring asymmetries due to spin-orbit and exchange interactions. Photoemission from a strained GaAs/GaAsP super lattice is used as the source of spin polarized electrons. Spin-orbit asymmetry is evaluated for Ir(100) single crystal at various energies. Subsequently, exchange asymmetry has been evaluated on 40 monolayer Fe deposited on Ir(100). This instrument proves to be useful in understanding structure and magnetism at surfaces. (C) 2016 AIP Publishing LLC.

Development of cell culture from caudal fin and heart of Tor tor (Hamilton-Buchanan)

Tor mahseer (Tor tor), possess high commercial and recreational value as they are potential game as well as food fish of India. Two cell culture systems were developed from fin and heart of T. tor (Hamilton-Buchanan). The explants excised aseptically from fingerling of T. tor were cultured in Leibovitz-15 (L-15) medium with 20% fetal bovine serum (FBS). Radiation of cells started after 72 hours and 48 hours of explant attachment from caudal fin and heart respectively. Confluent monolayer of cells with heterogeneous morphology around fin explants was observed after 7-10 days, where as a homogenous confluent layer of fibroblastic cells from heart explant was observed after 12-13 days. The establishment of cell culture systems from different organs and tissues of commercial important species would facilitates in vitro research.

Recent development of tip modification techniques in chemical force microscope

A review is given on the recent development of scanning probe microscope (SPM) tip modification techniques for chemical force microscope, including the preparation and application of SPM tip modified by self-assembled monolayer, atomic force microscope (AFM) tip modified by biological molecule, scanning tunneling microscope tip modified by electrochemical method, AFM tip modified by carbon nanotube.

Development of an in vitro multicellular tumor spheroid model using microencapsulation and its application in anticancer drug screening and testing

In this study, an in vitro multicellular tumor spheroid model was developed using microencapsulation, and the feasibility of using the microencapsulated. multicellular tumor spheroid (MMTS) to test the effect of chemotherapeutic drugs was investigated. Human MCF-7 breast cancer cells were encapsulated in alginate-poly-L-lysine-alginate (APA) microcapsules, and a single multicellular spheroid 150 mu m in diameter was formed in the microcapsule after 5 days of cultivation. The cell morphology, proliferation, and viability of the MMTS were characterized using phase contrast microscopy, BrdU-Iabeling, MTT stain, calcein AM/ED-2 stain, and H&E stain. It demonstrated that the MMTS was viable and that the proliferating cells were mainly localized to the periphery of the cell spheroid and the apoptotic cells were in the core. The MCF-7 MMTS was treated with mitomycin C (MC) at a concentration of 0.1, 1, or 10 times that of peak plasma concentration (ppc) for up to 72 h. The cytotoxicity was demonstrated. clearly by the reduction in cell spheroid size and the decrease in cell viability. The MMTS was further used to screen the anticancer effect of chemotherapeutic drugs, treated with MC, adriamycin (ADM) and 5-fluorouracil (5-FU) at concentrations of 0.1, 1, and 10 ppc for 24, 48, and 72 h. MCF-7 monolayer culture was used as control. Similar to monolayer culture, the cell viability of MMTS was reduced after treatment with anticancer drugs. However, the inhibition rate of cell viability in MMTS was much lower than that in monolayer culture. The MMTS was more resistant to anticancer drugs than monolayer culture. The inhibition rates of cell viability were 68.1%, 45.1%, and 46.8% in MMTS and 95.1%, 86.8%, and 91.6% in monolayer culture treated with MC, ADM, and 5-FU at 10 ppc for 72 h, respectively. MC showed the strongest cytotoxicity in both MMTS and monolayer, followed by 5-FU and ADM. It demonstrated that the MMTS has the potential to be a rapid and valid in vitro model to screen chemotherapeutic drugs with a feature to mimic in vivo three-dimensional (3-D) cell growth pattern.

Detection of Tetrodotoxins in Puffer Fish by a Self-Assembled Monolayer-Based Immunoassay and Comparison with Surface Plasmon Resonance, LC-MS/MS, and Mouse Bioassay

The increasing occurrence of puffer fish containing tetrodotoxin (TTX) in the Mediterranean could represent a major food safety risk for European consumers and threaten the fishing industry. The work presented herein describes the development of a new enzyme linked immunosorbent assay (mELISA) based on the immobilization of TTX through dithiol monolayers self-assembled on maleimide plates, which provides an ordered and oriented antigen immobilization and favors the antigen-antibody affinity interaction. The mELISA was found to have a limit of detection (LOD) of TTX of 0.23 mg/kg of puffer fish matrix. The mELISA and a surface plasmon resonance (SPR) immunosensor previously developed were employed to establish the cross-reactivity factors (CRFs) of 5,6,11-trideoxy-TTX, 5,11-deoxy-TTX, 11-nor-TTX-6-ol, and 5,6,11-trideoxy-4-anhydro-TTX, as well as to determine TTX equivalent contents in puffer fish samples. Results obtained by both immunochemical tools were correlated (R(2) = 0.977). The puffer fish samples were also analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the corresponding CRFs were applied to the individual TTX contents. Results provided by the immunochemical tools, when compared with those obtained by LC-MS/MS, showed a good degree of correlation (R(2) = 0.991 and 0.979 for mELISA and SPR, respectively). The mouse bioassay (MBA) slightly overestimated the CRF adjusted TTX content of samples when compared with the data obtained from the other techniques. The mELISA has been demonstrated to be fit for the purpose for screening samples in monitoring programs and in research activities.

The application of self-assembly monolayer technique into fabricating multilayer nanofilm

Self-Assembly Monolayer (SAM) technique, as a novel and developing technique for fabricating layer-by-layer nanofilm on substrates of various sizes, shapes and materials, has received more and more attention in the areas of light-emitting devices, nonlinear optical materials, conductive films, permselective gas membranes, sensors, modification of electrodes, resistance and printing technique. In comparison with other traditional methods, SAM technique has many significant advantages, including simple process, universality, formation with densely packed, well defined, highly ordered surfaces. This paper will give a review on the recent development in SAM technique.

Development of a novel micro-spray-assembly process for multilayer ultra-thin film formation

A novel micro-spray-assembly process and an automatic device to fabricate multilayer ultra-thin film are introduced. Employing self-assembly monolayer (SAM) technique, ultra-thin film can be assembled by utilizing the micro-spray-assembly device. The thickness and roughness of each monolayer can be controlled by varying various materials attributes, i.e., deposition time, ionic strength, pH value, molecular concentration and by selecting different manufacturing parameters of the automatic device such as spraying rate, size of micro-drop, N₂ flow rate, temperature of N₂ flow.

The self-assembled monolayer modification of electrodes - some recent advances in biological application

The modification of an electrode surface at the molecular level using the technique of depositing self-assembled monolayers (SAM) is a typical example of the techniques used in nanotechnology, from the process "bottom up", which is to create a nanostructure by successive additions of molecular or atomic entities on a surface. This article presents some recent advances in the field, with examples: the development of systems Sat hybridized with biomolecules, nanoparticles or nanotubes in bioelectronics, the use of switchable electrodes to study the adhesion and migration of biological cells , and the integration of molecular son in the SAM to recognize and allow the transduction of a biological response allowing the practice of electrochemistry in a complex biological environment.

Development of an Electrochemical Insulin Sensor Based on a High Affinity DNA Sequence Found in the Insulin-linked Polymorphic Region

The detection of pertinent biomarkers has the potential provide an early indication of disease progression before considerable damage has been incurred. A decrease in an individual’s sensitivity to insulin, which may be quantified as the ratio of insulin to glucose in the blood after a glucose pulse, has recently been reported as an early predictor of insulin-dependent diabetes mellitus. Routine measurement of insulin levels is therefore desirable in the care of diabetes-prone individuals. A rapid, simple, and reagentless method for insulin detection would allow for wide-spread screenings that provide earlier signs of diabetes onset. The aim of this thesis is to develop a folding-base electrochemical sensor for the detection of insulin. The sensor described herein consists of a DNA probe immobilized on a gold disc electrode via an alkanethiol linker and embedded in an alkanethiol self-assembled monolayer. The probe is labeled with a redox reporter, which readily transfers electrons to the gold electrode in the absence of insulin. In the presence of insulin, electron transfer is inhibited, presumably due to a binding-induced conformational or dynamic change in the DNA probe that significantly alters the electron-tunneling pathway. A 28-base segment of the insulin-linked polymorphic region that has been reported to bind insulin with high affinity serves as the capture element of the DNA probe. Three probe constructs that vary in their secondary structure and position of the redox label are evaluated for their utility as insulin-sensing elements on the electrochemical platform. The effects of probe modification on secondary structure are also evaluated using circular dichroism spectroscopy.